Differentially expressed proteins in cerulein-stimulated pancreatic acinar cells: implication for acute pancreatitis

The proteins expressed in pancreatic acinar cells during the initiation of acute pancreatitis may determine the severity of the disease. Cerulein pancreatitis is one of the best characterized models for acute pancreatitis. Present study aims to determine the differentially expressed proteins in cerulein-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. Rat pancreatic acinar AR42J cells were treated with 10(-8)M cerulein for 12h. The protein patterns separated by two-dimensional electrophoresis using pH gradients of 5-8 were compared between the cells treated without cerulein and those with cerulein. The changed proteins were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. As a result, 10 proteins (Orp150 protein, protein disulfide isomerase related protein, dnaK-type molecular chaperone hsp72-ps1, mitochondrial glutamate dehydrogenase, similar to chaperonin containing TCP-1 beta subunit, RuvB-like protein 1, heterogeneous nuclear ribonucleoprotein H1, aldehyde reductase 1, triosephosphate isomerase 1, peroxiredoxin 2) were up-regulated while four proteins (vasolin-containing protein, 78 kDa glucose-regulated protein precursor, heat shock protein 8, adenosylhomocysteinase) were down-regulated by cerulein in pancreatic acinar AR42J cells. These proteins are related to chaperone, cell defense mechanism against oxidative stress or DNA damage, anti-apoptosis and energy generation. The differentially expressed proteins by ceruein share their functional roles in pancreatic acinar cells, suggesting the possible involvement of oxidative stress, DNA damage, and anti-apoptosis in pathogenesis of acute pancreatitis. Proteins involved in cellular defense mechanism and energy production may protect pancreatic acinar cells during the development of pancreatitis.     

Ethanol induces secretion of oxidized proteins by pancreatic acinar cells

The pancreas is vulnerable to ethanol toxicity, but the pathogenesis of alcoholic pancreatitis is not fully defined. The intracellular oxidative balance and the characteristics of the secretion of isolated rat pancreatic acinar cells stimulated with the cholecystokinin analogue cerulein were assayed after acute oral ethanol (4 g/kg) load. Pancreatic acinar cells from ethanol-treated rats showed a significant (p < 0.02) lower content of total glutathione and protein sulfhydryls, and higher levels of oxidized glutathione (p < 0.03), malondialdehyde, and protein carbonyls (p < 0.05). Ethanol-intoxicated acinar cells showed a lower baseline amylase output compared to controls, with the difference being significantly exacerbated by cerulein stimulation. After cerulein, the release of protein carbonyls by ethanol-treated cells was significantly increased, whereas that of protein sulfhydryls was significantly decreased. In conclusion, ethanol oxidatively damages pancreatic acinar cells; cerulein stimulation is followed by a lower output of amylase and by a higher release of oxidized proteins by pancreatic acinar cells from ethanol-treated rats. These findings may account for the decreased exocrine function, intraductular plug formation, and protein precipitation in alcoholic pancreatitis.     

A rat model reproducing key pathological responses of alcoholic chronic pancreatitis

Although alcohol abuse is the major cause of chronic pancreatitis, the pathogenesis of alcoholic chronic pancreatitis (ACP) remains obscure. A critical obstacle to understanding the mechanism of ACP is lack of animal models. Our objective was to develop one such model. Rats were pair-fed for 8 wk ethanol or control Lieber-DeCarli liquid diet. For the last 2 wk, they received cyclosporin A (CsA; 20 mg/kg once daily) or vehicle. After 1 wk on CsA, one episode of acute pancreatitis was induced by four 20 microg/kg injections of cerulein (Cer); controls received saline. Pancreas was analyzed 1 wk after the acute pancreatitis. CsA or Cer treatments alone did not result in pancreatic injury in either control (C)- or ethanol (E)-fed rats. We found, however, that alcohol dramatically aggravated pathological effect of the combined CsA+Cer treatment on pancreas, resulting in massive loss of acinar cells, persistent inflammatory infiltration, and fibrosis. Macrophages were prominent in the inflammatory infiltrate. Compared with control-fed C+CsA+Cer rats, their ethanol-fed E+CsA+Cer counterparts showed marked increases in pancreatic NF-kappaB activation and cytokine/chemokine mRNA expression, collagen and fibronectin, the expression and activities of matrix metalloproteinase-2 and -9, and activation of pancreatic stellate cells. Thus we have developed a model of alcohol-mediated postacute pancreatitis that reproduces three key responses of human ACP: loss of parenchyma, sustained inflammation, and fibrosis. The results indicate that alcohol impairs recovery from acute pancreatitis, suggesting a mechanism by which alcohol sensitizes pancreas to chronic injury.     

Fibroblast growth factor 21 alleviates acute pancreatitis via activation of the Sirt1‐autophagy signalling pathway

Fibroblast growth factor 21 (FGF21), a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity, alleviates the process of acute pancreatitis (AP). However, its mechanism remains elusive. The pathological and physiological characteristics of FGF21 are observed in both patients with AP and cerulein‐induced AP models, and the mechanisms of FGF21 in response to AP are investigated by evaluating the impact of autophagy in FGF21‐treated mice and cultured pancreatic cells. Circulating levels of FGF21 significantly increase in both AP patients and cerulein‐induced AP mice, which is accompanied by the change of pathology in pancreatic injury. Replenishment of FGF21 distinctly reverses cerulein‐induced pancreatic injury and improves cerulein‐induced autophagy damage in vivo and in vitro. Mechanically, FGF21 acts on pancreatic acinar cells to up‐regulate Sirtuin‐1 (Sirt1) expression, which in turn repairs impaired autophagy and removes damaged organs. In addition, blockage of Sirt1 accelerates cerulein‐induced pancreatic injury and weakens the regulative effect in FGF21‐activated autophagy in mice. These results showed that FGF21 protects against cerulein‐induced AP by activation of Sirtuin‐1‐autophagy axis.     

Proteome analysis of rat pancreatic acinar cells: implication for cerulein-induced acute pancreatitis

Cerulein pancreatitis was shown to be one of the best characterized models for acute pancreatitis. High doses of cerulein induce a dysregulation of the digestive enzyme production and cytoplasmic vacuolization and the death of acinar cells, edema formation, and an infiltration of inflammatory cells into the pancreas, which are similar symptoms shown in human acute pancreatitis. The present study aims to determine the differentially expressed proteins in cerulein-treated pancreatic acinar cells as an in vitro model for acute pancreatitis. Pancreatic acinar AR42J cells were treated with 10(-8) M cerulein for 24 h. The changed protein patterns separated by two-dimensional electrophoresis using pH gradients of 5-8 were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis of the peptide digests. Five differentially expressed proteins (heat shock protein 90, mitochondrial ATP synthase beta chain precursor, tubulin beta chain, 3-mercaptopyruvate sulfurtransferase, mitochondrial ATP synthase subunit D) were identified in cerulein-treated AR42J cells. These proteins are related to cellular stress such as reactive oxygen species, cytoskeletal function, and cell signaling. In conclusion, the differentially expressed proteins will provide valuable information to understand the pathophysiologic mechanism of acute pancreatitis and may be useful for prognostic indices of acute pancreatitis.     

Diphenyleneiodonium suppresses apoptosis in cerulein-stimulated pancreatic acinar cells

NADPH oxidase has been considered a major source of reactive oxygen species in phagocytic and non-phagocytic cells. Apoptosis linked to oxidative stress has been implicated in pancreatitis. Recently, we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox, and p67phox are constitutively expressed in pancreatic acinar cells, which are activated by cerulein, a cholecystokinin analogue. Cerulein induces an acute and edematous form of pancreatitis. We investigated whether inhibition of NADPH oxidase by diphenyleneiodonium suppresses the production of reactive oxygen species and apoptosis by determining viable cell numbers, DNA fragmentation, TUNEL staining, caspase-3 activity, and the expression of apoptosis-inducing factor in pancreatic acinar AR42J cells stimulated with cerulein. Inhibition on NADPH oxidase by diphenyleneiodonium was assessed by the alterations in NADPH oxidase activity and translocation of the cytosolic subunits p67phox and p47phox to the membrane. Intracellular Ca2+ level was monitored to investigate the relationship between NADPH oxidase and Ca2+ in cells stimulated with cerulein. As a result, cerulein induced the activation of NADPH, increased production of reactive oxygen species, and apoptotic indices determined by the expression of apoptosis-inducing factor, caspase-3 activation, TUNEL staining, DNA fragmentation, and cell viability. Treatment with DPI inhibited cerulein-induced activation of NADPH oxidase, the production of reactive oxygen species, and apoptosis, but not the increase of intracellular Ca2+ levels in pancreatic acinar cells. These results demonstrate that the cerulein-induced increase in intracellular Ca2+ level may be an upstream event of NADPH oxidase activation. Diphenyleneiodonium, an NADPH oxidase inhibitor, inhibits the expression of apoptosis-inducing factor and caspase-3 activation, and thus apoptosis in pancreatic acinar cells.     

2',4',6'-Tris(methoxymethoxy) chalcone (TMMC) attenuates the severity of cerulein-induced acute pancreatitis and associated lung injury

Acute pancreatitis (AP) is an inflammatory disease involving acinar cell injury and rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. 2',4',6'-Tris (methoxymethoxy) chalcone (TMMC), a synthetic chalcone derivative, displays potent anti-inflammatory effects. Therefore, we aimed to investigate whether TMMC might affect the severity of AP and pancreatitis-associated lung injury in mice. We used the cerulein hyperstimulation model of AP. Severity of pancreatitis was determined in cerulein-injected mice by histological analysis and neutrophil sequestration. The pretreatment of mice with TMMC reduced the severity of AP and pancreatitis-associated lung injury and inhibited several biochemical parameters (activity of amylase, lipase, trypsin, trypsinogen, and myeloperoxidase and production of proinflammatory cytokines). In addition, TMMC inhibited pancreatic acinar cell death and production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 by inhibiting NF-κB and extracellular signal-regulated protein kinase 1/2 (ERK1/2) activation. Neutralizing antibodies for TNF-α, IL-1β, and IL-6 inhibited cerulein-induced cell death in isolated pancreatic acinar cells. Moreover, pharmacological blockade of NF-κB/ERK1/2 reduced acinar cell death and production of TNF-α, IL-1β, and IL-6 in isolated pancreatic acinar cells. In addition, posttreatment of mice with TMMC showed reduced severity of AP and lung injury. Our results suggest that TMMC may reduce the complications associated with pancreatitis.     

Protective Effects of Hydrogen Gas on Experimental Acute Pancreatitis

Acute pancreatitis (AP) is an inflammatory disease mediated by damage to acinar cells and pancreatic inflammation. In patients with AP, subsequent systemic inflammatory responses and multiple organs dysfunction commonly occur. Interactions between cytokines and oxidative stress greatly contribute to the amplification of uncontrolled inflammatory responses. Molecular hydrogen (H₂) is a potent free radical scavenger that not only ameliorates oxidative stress but also lowers cytokine levels. The aim of the present study was to investigate the protective effects of H₂ gas on AP both in vitro and in vivo. For the in vitro assessment, AR42J cells were treated with cerulein and then incubated in H₂-rich or normal medium for 24 h, and for the in vivo experiment, AP was induced through a retrograde infusion of 5% sodium taurocholate into the pancreatobiliary duct (0.1 mL/100 g body weight). Wistar rats were treated with inhaled air or 2% H₂ gas and sacrificed 12 h following the induction of pancreatitis. Specimens were collected and processed to measure the amylase and lipase activity levels; the myeloperoxidase activity and production levels; the cytokine mRNA expression levels; the 8-hydroxydeoxyguanosine, malondialdehyde, and glutathione levels; and the cell survival rate. Histological examinations and immunohistochemical analyses were then conducted. The results revealed significant reductions in inflammation and oxidative stress both in vitro and in vivo. Furthermore, the beneficial effects of H₂ gas were associated with reductions in AR42J cell and pancreatic tissue damage. In conclusion, our results suggest that H₂ gas is capable of ameliorating damage to the pancreas and AR42J cells and that H₂ exerts protective effects both in vitro and in vivo on subjects with AP. Thus, the results obtained indicate that this gas may represent a novel therapy agent in the management of AP.     

Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore conclude that expression of WT or mutant human PRSS1 in murine acinar cells induces apoptosis and is sufficient to promote spontaneous pancreatitis, which is enhanced in response to cellular insult.     

L- 精氨酸和雨蛙素诱导急性胰腺炎模型的对比研究

目的:研究L-精氨酸和雨蛙素分别诱导SD大鼠急性胰腺炎(AP)模型的差异,为进一步研究急性胰腺炎提供可靠模型。方法:L-精氨酸采用3次腹腔注射,间隔1 h,雨蛙素采用7次腹腔注射,间隔1 h诱导急性胰腺炎模型。碘-淀粉比色法检测血清淀粉酶水平,血清脂肪酶测定试剂盒检测脂肪酶活性,胰腺组织切片观察组织的破坏情况,TUNEL法检测腺泡细胞凋亡。结果:①L-精氨酸诱导的大鼠模型血清淀粉酶和脂肪酶水平在诱导成功后6 h即显著升高,蛙皮素诱导的大鼠模型在12 h显著升高,与正常对照组比较均有统计学差异(P<0.05),提示急性胰腺炎建模成功。②L-精氨酸诱导的模型中胰腺组织结构破坏,有大片出血坏死灶、大量炎细胞浸润;而蛙皮素诱导的模型组织腺泡、间质水肿,炎性细胞浸润,少量散在出血坏死灶,血管变化常不明显,渗液清亮。结论:L-精氨酸和雨蛙素均能诱导SD大鼠急性胰腺炎模型,L-精氨酸诱导重症急性胰腺炎,雨蛙素诱导轻型急性胰腺炎,是研究急性胰腺炎的良好模型。     

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis

BackgroundDuring pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence.MethodsWe examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis.ResultsPancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin.ConclusionOur findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.     

Tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone initiates and enhances pancreatitis responses

Clinical studies indicate that cigarette smoking increases the risk for developing acute pancreatitis. The nicotine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a major cigarette smoke toxin. We hypothesized that NNK could sensitize to pancreatitis and examined its effects in isolated rat pancreatic acini and in vivo. In acini, 100 nM NNK caused three- and fivefold activation of trypsinogen and chymotrypsinogen, respectively, above control. Furthermore, NNK pretreatment in acini enhanced zymogen activation in a cerulein pancreatitis model. The long-term effects of NNK were examined in vivo after intraperitoneal injection of NNK (100 mg/kg body wt) three times weekly for 2 wk. NNK alone caused zymogen activation (6-fold for trypsinogen and 2-fold for chymotrypsinogen vs. control), vacuolization, pyknotic nuclei, and edema. This NNK pretreatment followed by treatment with cerulein (40 μg/kg) for 1 h to induce early pancreatitis responses enhanced trypsinogen and chymotrypsinogen activation, as well as other parameters of pancreatitis, compared with cerulein alone. Potential targets of NNK include nicotinic acetylcholine receptors and β-adrenergic receptors; mRNA for both receptor types was detected in acinar cell preparations. Studies with pharmacological inhibitors of these receptors indicate that NNK can mediate acinar cell responses through an nonneuronal α(7)-nicotinic acetylcholine receptor (α(7)-nAChR). These studies suggest that prolonged exposure to this tobacco toxin can cause pancreatitis and sensitize to disease. Therapies targeting NNK-mediated pathways may prove useful in treatment of smoking-related pancreatitis.     

Recruitment and activation of pancreatic stellate cells from the bone marrow in pancreatic cancer: a model of tumor-host interaction

Background and Aims

Chronic pancreatitis and pancreatic cancer are characterised by extensive stellate cell mediated fibrosis, and current therapeutic development includes targeting pancreatic cancer stroma and tumor-host interactions. Recent evidence has suggested that circulating bone marrow derived stem cells (BMDC) contribute to solid organs. We aimed to define the role of circulating haematopoietic cells in the normal and diseased pancreas.

Methods

Whole bone marrow was harvested from male β-actin-EGFP donor mice and transplanted into irradiated female recipient C57/BL6 mice. Chronic pancreatitis was induced with repeat injections of caerulein, while carcinogenesis was induced with an intrapancreatic injection of dimethylbenzanthracene (DMBA). Phenotype of engrafted donor-derived cells within the pancreas was assessed by immunohistochemistry, immunofluorescence and in situ hybridisation.

Results

GFP positive cells were visible in the exocrine pancreatic epithelia from 3 months post transplantation. These exhibited acinar morphology and were positive for amylase and peanut agglutinin. Mice administered caerulein developed chronic pancreatitis while DMBA mice exhibited precursor lesions and pancreatic cancer. No acinar cells were identified to be donor-derived upon cessation of cerulein treatment, however rare occurrences of bone marrow-derived acinar cells were observed during pancreatic regeneration. Increased recruitment of BMDC was observed within the desmoplastic stroma, contributing to the activated pancreatic stellate cell (PaSC) population in both diseases. Expression of stellate cell markers CELSR3, PBX1 and GFAP was observed in BMD cancer-associated PaSCs, however cancer-associated, but not pancreatitis-associated BMD PaSCs, expressed the cancer PaSC specific marker CELSR3.

Conclusions

This study demonstrates that BMDC can incorporate into the pancreas and adopt the differentiated state of the exocrine compartment. BMDC that contribute to the activated PaSC population in chronic pancreatitis and pancreatic cancer have different phenotypes, and may play important roles in these diseases. Further, bone marrow transplantation may provide a useful model for the study of tumor-host interactions in cancer and pancreatitis.     

Raf-1 Kinase Inhibitory Protein (RKIP) Mediates Ethanol-induced Sensitization of Secretagogue Signaling in Pancreatic Acinar Cells

Excessive alcohol consumption is associated with most cases of chronic pancreatitis, a progressive necrotizing inflammatory disease that can result in pancreatic insufficiency due to acinar atrophy and fibrosis and an increased risk of pancreatic cancer. At a cellular level acute alcohol exposure can sensitize pancreatic acinar cells to secretagogue stimulation, resulting in dysregulation of intracellular Ca²⁺ homeostasis and premature digestive enzyme activation; however, the molecular mechanisms by which ethanol exerts these toxic effects have remained undefined. In this study we identify Raf-1 kinase inhibitory protein as an essential mediator of ethanol-induced sensitization of cholecystokinin- and carbachol-regulated Ca²⁺ signaling in pancreatic acinar cells. We show that exposure of rodent acinar cells to ethanol induces protein kinase C-dependent Raf-1 kinase inhibitory protein phosphorylation, sensitization of cholecystokinin-stimulated Ca²⁺ signaling, and potentiation of both basal and cholecystokinin-stimulated extracellular signal-regulated kinase activation. Furthermore, we show that either suppression of Raf-1 kinase inhibitory protein expression using short hairpin RNA or gene ablation prevented the sensitizing effects of ethanol on cholecystokinin- and carbachol-stimulated Ca²⁺ signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of Raf-1 inhibitory protein expression may have future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.